Cells screening method

ABSTRACT

Provided are a method and means permitting the simultaneous measurement of the reactive properties of more than 10,000 of antigen-stimulated lymphocytes being held on a chip. A microwell array comprises multiple wells capturing single cells and a coating layer on a principal surface around the wells containing a substance capable of binding to a substance produced by the cells in the wells. A method for screening a target cell comprises: causing specimen cells and a cell culture broth to be contained in the wells of the microwell array; culturing the cells in a state permitting the diffusion of substances from the wells into the coating layer; feeding a label substance binding specifically to a substance produced by a target cell onto the coating layer; and detecting the substance produced by the target cell by the label substance binding around the well to specify the target cell.

CROSS-REFERENCE TO A RELATED PATENT APPLICATION

The present application claims priority under Japanese PatentApplication No. 2007-201493 filed on Aug. 2, 2007, the entire contentsof which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for screening cells, and to amicrowell array employed in this method. More particularly, the presentinvention relates to a method for screening immune cells such asspecific immunoglobulin-producing cells and specific cytokine-producingcells, and to a microwell array employed in this method.

BACKGROUND ART

Conventionally, antigen-specific antibody-producing hybridomas have beenprepared to produce monoclonal antibodies. In the conventional method ofpreparing hybridomas, hybridomas are prepared, after which hybridomaclones producing antigen-specific antibodies are screened. However, thepreparation of hybridomas is not efficient. That is, not all Blymphocytes become hybridomas; only some of the B lymphocytes in whichcell fusion with a myeloma has occurred become hybridomas. Even when ahybridoma is produced with a spleen cell that has been stimulated withan antigen, not just antigen-specific antibody-producing hybridomas areproduced; most of the hybridomas that are produced either produceunrelated antibodies or do not produce antibodies at all.

For example, when looking for a hybridoma producing a target antibody bythe conventional method, spleen cells taken from an immunized mouse aresubjected to cellular fusion with myelomas and sown into about ten96-well plates. More could be sown if all the cells were used, but thereare time limits when a single person is doing the screening, and thosethat remain are stored by freezing or the like. Normally, hybridomasgrow in about 500 wells when using this method.

The hybridomas in the 500 wells do not all proliferate at the samespeed; some grow quickly and others grow slowly. Accordingly, it isimpossible to check the growth of all 500 simultaneously. First, a checkmust be made under a microscope as to which wells have produced cellgrowth, and whether the number of cells has increased sufficiently tocheck for antibodies. Subsequently, cell supernatant is collected fromsuitable wells, and a check is made for the production ofantigen-specific antibodies. It is necessary to perform the cell checkand cell supernatant check extremely rapidly. This is because hybridomasgrow steadily and proliferate excessively if left alone, depleting thenutrients in the medium and dying out. Accordingly, screening must becompleted before the desired hybridomas die.

Further, once a well is found in which a target hybridoma is growing,frequently there will be hybridomas producing other antibodies growingin the well in addition to the hybridoma producing the target antibody.Further, since hybridomas drop their own chromosomes while growing,there are also cases where a hybridoma that has been producing anantibody ends up losing the chromosome with the antibody and becomesunable to produce the antibody. The growth of such cells is often morerapid than that of hybridomas that are producing antibody, and most ofthe cells that are cultured when left alone end up becomingnon-antibody-producing cells. Accordingly, when a well is discovered inwhich a desired hybridoma is growing, the cells in that well areimmediately reseeded one cell per well in a 96-well plate (criticaldilution method), and screening is conducted again for desiredantibody-producing hybridomas (secondary screening). Once a targetedhybridoma has been detected, it is necessary to rapidly proceed throughsecondary screening before the state of the cell deteriorates.

As set forth above, since screening is sometimes conducted with justsome of the hybridomas that are prepared, without screening them all, itbecomes difficult to obtain low-frequency antigen-specificantibody-producing hybridomas.

More specifically, in the case of human antigen-specific antibodies,there exists a method of screening cells producing antigen-specificantibodies in strains developed by transforming peripheral B lymphocyteswith EB virus (Non-patent Document 1). In this method, since thefrequency of the lymphocyte cell strains established is low, theprobability of obtaining an antigen-specific antibody-producing Blymphocyte cell strain is extremely low. Further, it takes about a monthto establish a cell strain. Still further, the B lymphocyte strains thatare established produce only small quantities of antibody. Althoughhybridomas can be prepared for mice, no system for producing hybridomaswith good efficiency has been developed for humans.

Hybridomas can be prepared for mice. Conventionally, to produce ahybridoma, a mouse is immunized with an antigen, the spleen or lymphnodes of the mouse are removed, lymphocytes are prepared, about 10⁸ ofthe lymphocytes prepared and about 10⁷ myeloma cells are fused usingpolyethylene glycol or by subjecting them to a voltage, they arecultured in a selection medium such as HAT, the hybridomas that grow arescreened by ELISA, flow cytometry, or the like to determine whether ornot they are producing the antigen-specific antibody, and theantigen-specific antibody-producing hybridomas are selected (Non-patentDocuments 2 and 3). When employing this method, hybridomas grow in 300to 400 wells. Of these, hybridomas producing antigen-specific antibodiesgrow in only several percent of the wells. This number varies with theantigen employed, but, it is difficult to prepare hybridomas by thismethod when the frequency of the antigen-specific antibody-producing Blymphocyte is low.

Accordingly, the present inventors examined methods of convenientlyselecting lymphocytes reacting specifically with prescribed antigens inthe form of both antigen-specific lymphocytes of relatively highfrequency and antigen-specific lymphocytes of low frequency, andpreparing antigen-specific antibody-producing hybridomas from theantigen-specific B lymphocytes that were selected. They then devised amethod for preparing antigen-specific antibody-producing hybridomas byculturing selected antigen-specific B lymphocytes and fusing theantigen-specific B lymphocytes grown by culturing with myeloma cells toprepare hybridomas, and applied for a patent (Patent Document 1).

Attempts have also been made to specify and select individual cells, anduse the cells that have been selected. For example, the separatedetection of individual antigen specificity, the recovery of a singledetected antigen-specific lymphocyte, and the use of the singleantigen-specific lymphocyte recovered to prepare an antibody, forexample, have been examined (Patent Documents 2 and 3).

-   [Patent Document 1] WO2004/087911-   [Patent Document 2] Japanese Patent Un-examined Publication    2004-173681-   [Patent Document 3] Japanese Patent Un-examined Publication    2004-187676-   [Non-patent Document 1] “Methods of Detecting Lymphocyte Functions    (Version 5)”, Junichi YANO, Michio FUJIWARA, eds., Chugai Igakusha    (1994), “Use of EB virus transform B cells for preparation of human    monoclonal antibody”, Fumio MIZUNO, Toshiro OHSATO, pp 381-391.-   [Non-patent Document 2] “Methods of Detecting Lymphocyte Functions    (Version 5)”, Junichi YANO, Michio FUJIWARA, eds., Chugai Igakusha    (1994), “Preparation of monoclonal antibody with B cell hybridomas”,    Hideo NARIUCHI, pp 574-576-   [Non-patent Document 3] Monoclonal antibodies in “Antibodies: A    Laboratory Manual” by Ed Harlow and David Lane, Cold Spring Harbor    Laboratory, Cold Spring Harbor, N.Y., pp 139-pp 244, 1988-   [Non-patent Document 4] In vitro antibody production. In Current    Protocols in Immunology. Edited by J. E. Coligan et al., John Wiley    & Sons (New York), p. 3.8.1-3.8.16, 1991.-   [Non-patent Document 5] Babcook, J. S., Leslie, K. B., Olsen, O. A.,    Salmon, R. A., Schrader, J. W. A novel strategy for generating    monoclonal antibodies from single, isolated lymphocytes producing    antibodies of defined specificities. Proc Natl Acad Sci USA, 93:    7843-7848, 1996.-   [Non-patent Document 6] Measurement of polyclonal immunoglobulin    synthesis using the ELISPOT assay. In Current Protocols in    Immunology. Edited by J. E. Coligan et al., John Wiley & Sons (New    York), p. 7.14.1-7.14.7, 1991.-   [Non-patent Document 7] Assays for antibody production. In Current    Protocols in Immunology. Edited by J. E. Coligan et al., John Wiley    & Sons (New York), p. 2.1.1-2.1.22, 1991.-   [Non-patent Document 8] J. C. Love, J. L. Ronan, G. M.    Grotenbreg, A. G. van der Veen, H. L. Ploegh, A microengraving    method for rapid selection of single cells producing    antigen-specific antibodies. Nature Biotechnology, 24: 703-707,    2006.

The entire contents of Patent Documents 1-3 and Non-patent Documents 1-8are hereby incorporated by reference.

In the above-described conventional methods, the separate detection ofthe antigen specificity of individual lymphocytes and the recovery ofthe antigen-specific lymphocytes that are detected are conductedmanually. In Patent Documents 2 and 3, it is stated that thespecification of cells at the cellular level has been confirmed, andthat it is also possible to recover the specified cells. However, theactual detection of those lymphocytes that have specifically reactedwith an antigen from among numerous lymphocytes is difficult.

As an example of a detection method, the fact that the calcium ionconcentration rises in lymphocytes that have reacted with an antigen isexploited, this change in calcium ion concentration is detected byfluorescence, and antigen-specific lymphocytes are specified. However,depending on the type of cell (lymphocyte), the method of generatingfluorescence, the intensity thereof, and the like may differ. There arelymphocytes in which the calcium ion concentration rises immediatelyupon antigen stimulation, resulting in an increase in the intensity offluorescence. However, there are also cells in which the calcium ionconcentration rises only after a certain period has elapsed followingantigen stimulation, resulting in a rise in the intensity offluorescence. Further, it is necessary to measure nearly simultaneouslyand in one shot the intensity of fluorescence of about 10,000 to 200,000lymphocytes being held on the surface of a several centimeter squaretube. Still further, since this fluorescence arises in individual cells,the intensity of the fluorescence is low, requiring highly sensitivefluorescence detection.

Thus far, there exists neither a method nor a device capable ofsimultaneously measuring the intensity of numerous sources of weakfluorescence gathered in highly concentrated fashion on the surface of atube.

One conventional device is the laser scanning cytometer. With a laserscanning cytometer, it is possible to measure the concentration ofcalcium in several hundred thousand individual cells. However, a singlescan requires a lengthy 10 minutes or more, precluding real-timedetection in lymphocytes the fluorescence intensity of which changesseveral minutes after antigen stimulation.

A fluorescent microscope with projector is comprised of an ordinaryfluorescent microscope combined with a projection device. A fluorescentmicroscope with projector presents no problem in terms of speed.However, as an ordinary microscope, the scope of projection is narrow,making it possible to detect fluorescence in only about 1,000 cells at atime. It is impossible to detect fluorescence in several tens ofthousands to several hundred thousand cells at a time.

A cell chip detector based on a DNA microarray scanner is a device thathas been developed to detect fluorescence in cells arrayed in cellchips, and can detect fluorescence inside or outside cells. However, itis a system that excites a fluorescent dye with a laser and detects theexcitation light. Thus, in detection systems that track changes overtime, the line scanning rate of the laser is slow, precluding thesimultaneous detection of numerous cell regions.

In the methods described in above-cited Patent Documents 2 and 3, thereaction detection system for detecting cells reacting specifically toan antigen is required to have the ability to capture in real timechanges in calcium over time within individual cells and the ability todetect individual cells that are present in only small number (reactonly slightly) among large numbers of cells, necessitating the abilityto separately and simultaneously analyze large numbers of cells inparallel.

Accordingly the object of the present invention is to provide a methodpermitting the simultaneous measurement of the states of a large numberof cells, exceeding 10,000 and desirably exceeding 100,000, being heldon a chip, such as the reactive properties of antigen-stimulatedlymphocytes, and the separate determination of the states of individualcells.

B lymphocytes express a single antibody on the cell surface. When apathogen (antigen) or the like invades the body, the antigen binds tothe antibody on the cell surface, activating the cell and causing it toproliferate and differentiate. Finally, it differentiates into anantibody-secreting cell. There exist already numerous methods ofspecifying antibody-secreting cells. Typical methods of specifyingsingle antibody-secreting cells are the plaque method and theEnzyme-Linked Immunospot (ELISPOT) method.

The plaque method is a method of specifying antigen-specificantibody-secreting cells in which hemolysis induced by bindingantibodies to erythrocytes that have been labeled with the antigenssurrounding antibody-secreting cells is employed as an indicator(Non-patent Document 4). A method that detects antigen-specificantibody-secreting cells by the plaque method and recovers the antibodygene has already been developed (Non-patent Document 5).

The ELISPOT method is a method of inoculating cells on a plate that hasbeen coated with antigen, causing the antibody that is secreted byantibody-secreting cells to bind to the antigen around the cells, anddetecting this with enzyme-labeled anti-Ig antibody or the like(Non-patent Document 6). With the ELISPOT method, in the course ofdetecting the binding of antigen-specific antibody around cells withenzyme-labeled anti-Ig antibody, the cells themselves get washed away,precluding recovery of the antigen-specific antibody-secreting cells.

Hybridomas are antibody-secreting cells. However, in the course ofscreening hybridomas, enzyme-linked immunosorbent assay (ELISA) iscommonly employed (Non-patent Document 7). Recently, this method hasbeen further developed; a method has been reported whereby individualhybridomas are cultured in small wells, and the antibody that issecreted in each well is detected by an antigen or the like labeled witha fluorescent label to detect antigen-specific antibody-secreting cells(Non-patent Document 8).

To achieve the above-stated object of the present invention, the presentinventors conducted extensive research into providing a method fordetecting immune cells, such as new antigen-specific antibody-secretingcells, that was completely different from the methods described inNon-patent Documents 4 to 8. The present invention was devised on thatbasis.

DISCLOSURE OF THE INVENTION

The present invention, devised to solve the above-stated problems, isset forth below:

[1] A microwell array, comprising multiple wells on one of the principalsurfaces of a base member, the wells being of a size permitting theentry of only a single cell into each well, characterized in that:

a coating layer of a substance capable of binding to at least part of asubstance produced by at least a portion of the cells contained in thewells is present on at least part of the principal surface at leastaround the wells.

[2] The microwell array according to [1] which is used:

to contain cells and cell culture solution in at least a portion of thewells thereof;

to immerse the coating layer and the wells in the culture solution andculture the cells in a state permitting the diffusion of a substancecontained in the culture solution into the coating layer; and

once the culture solution has been removed following culturing, todetect the presence or absence of binding between a substance that hasbeen produced by at least a portion of the cells contained in the wellsand the substance in the coating layer.

[3] The microwell array according to [1] or [2], wherein at least aportion of the cells contained in the wells are immunoglobulin-producingcells or cytokine-producing cells.

[4] The microwell array according to [1] or [2], wherein at least aportion of the cells contained in the wells are immunoglobulin-producingcells, and the substance having the ability to bind to at least aportion of a substance produced by the immunoglobulin-producing cells isan anti-immunoglobulin antibody or antigen.[5] The microwell array according to [4], wherein the detection of thepresence or absence of the binding is conducted using an antigen or anantibody to the immunoglobulin that is produced.[6] The microwell array according to [1] or [2], wherein at least aportion of the cells contained in the wells are cytokine-producingcells, and the substance having the ability to bind to at least aportion of the substance produced by the cytokine-producing cells is ananti-cytokine antibody or a cytokine receptor.[7] The microwell array according to [6], wherein the detection of thepresence or absence of the binding is conducted using a cytokinereceptor or an antibody to the cytokine produced.[8] The microwell array according to [1] or [2], wherein at least aportion of the cells contained in the wells are immunoglobulin-producingcells, and the substance having the ability to bind to at least aportion of the substance produced by the immunoglobulin-producing cellsis a cytokine receptor or a receptor.[9] The microwell array according to [8], wherein the detection of thepresence or absence of the binding is conducted using a cytokine or aligand.[10] The microwell array according to any one of [1] to [9], wherein theimmunoglobulin-producing cells and the cytokine-producing cells arenatural cells or hybridomas.[11] The microwell array according to any one of [1] to [10], whereinthe base member is plate-shaped.[12] The microwell array according to any one of [1] to [11], wherein atleast a portion of the principal surface on which is not present acoating layer of the substance having the ability to bind to at least aportion of the substance produced by the cells stored in the wells iscoated with a blocking agent.[13] A method of screening for a target cell, comprising:

causing specimen cells and a cell culture broth to be contained in atleast a portion of the wells of a microwell array comprising multiplewells, each well being of a size permitting the entry of only a singlecell therein, on one of the principal surfaces of a base member, andcomprising a coating layer of a substance having the ability to bind toat least a portion of a substance produced by a target cell on at leasta portion of the principal surface at least around the wells;

immersing the coating layer and the wells in the culture broth andculturing the cells in a state permitting the diffusion of substances inthe culture broth from the wells into the coating layer;

after optionally removing the culture broth, feeding a label substancebinding specifically to a substance produced by a target cell presentamong the specimen cells onto the coating layer; and

detecting the substance produced by the target cell that has bound tothe substance in the coating layer by means of the label substance tospecify the target cell.

-   [14] The screening method according to [13], wherein the cells    present among the specimen cells include cells that have been    stimulated with a desired antigen in advance and are in a state    capable of producing a substance.    [15] The screening method according to [13] or [14], wherein the    specimen cells include immunoglobulin-producing cells or    cytokine-producing cells.    [16] The screening method according to [13] or [14], wherein the    specimen cells include immunoglobulin-producing cells, a substance    having the ability to bind to at least a portion of the substance    produced by the target cell is an anti-immunoglobulin antibody or    antigen, and the target cell is an antigen-specific    immunoglobulin-producing cell.    [17] The screening method according to [16], wherein the detection    of the presence or absence of the binding is conducted using antigen    or an antibody to the immunoglobulin that is produced.    [18] The screening method according to [13] or [14], wherein the    specimen cells include immunoglobulin-producing cells, the substance    having the ability to bind to at least a portion of the substance    produced by the target cell is a cytokine receptor or receptor, and    the target cell is an antigen-specific immunoglobulin-producing    cell.    [19] The screening method according to [18], wherein the detection    of the presence or absence of the binding is conducted using a    cytokine or a ligand.    [20] The screening method according to [13] or [14], wherein the    specimen cells include cytokine-producing cells, the substance    having the ability to bind to at least a portion of the substance    produced by the target cell is an anti-cytokine antibody or a    cytokine receptor, and the target cell is an antigen-specific    cytokine-producing cell.    [21] The screening method according to [20], wherein the detection    of the presence or absence of the binding is conducted using an    antibody to the cytokine produced or a cytokine receptor.    [22] The screening method according to any one of [13] to [21],    wherein the immunoglobulin-producing cells and cytokine-producing    cells are natural cells, hybridomas, or cell strains.    [23] A method for producing a target cell, comprising recovering    from a well a target cell specified by the screening method in    accordance with any one of [13] to [22].    [24] The production method in accordance with [23], wherein the    target cell is a specific immunoglobulin-producing cell or specific    cytokine-producing cell.

The present invention provides a method and a means permitting thesimultaneous measurement of the states of a large number of cells,exceeding 10,000, for example, and desirably exceeding 100,000, beingheld on a chip, such as the reactive properties of antigen-stimulatedlymphocytes, and the separate determination of the states of individualcells.

BEST MODES OF CARRYING OUT THE INVENTION

[The Microwell Array]

The microwell array of the present invention is a microwell array havingmultiple wells on one of the principal surfaces of a base member, thewells being of a size permitting the entry of only a single cell. Amicrowell array having such a structure may be employed in the formdescribed in Patent Documents 1 to 3, for example.

Multiple microwells are disposed in equally spaced rows and columns on amicrowell array chip.

Neither the shape nor the size of the microwells is specificallylimited. However, for example, the shape of the microwell can becylindrical. It can also be noncylindrical, such as a polyhedroncomprised of multiple faces (for example, a parallelepiped, hexagonalcolumn, or octagonal column), an inverted cone, an inverted pyramid(inverted triangular pyramid, inverted square pyramid, invertedpentagonal pyramid, inverted hexagonal pyramid, or an inverted polygonalpyramid with seven or more angles), or have a shape combining two ormore of these shapes. For example, it may be partly cylindrical, withthe remainder having the shape of an inverted cone. In the case of aninverted conical or an inverted pyramidal shape, the mouth of themicrowell is on the bottom. However, the shape may be one in which aportion of the top of an inverted cone or inverted pyramid is cut off(in which case the bottom of the microwell is flat). For conical andparallelepiped shapes, the bottom of the microwell is normally flat, butcurved surfaces (convex or concave) are also possible. The reason thebottom of the microwell is made curved is the same as for shapesconsisting of an inverted cone or inverted pyramid with a portion of thetop cut off.

For a cylindrically-shaped microwell, the dimensions can be, forexample, a diameter of 3 to 100 micrometers. When the organic cell is aB lymphocyte, the diameter is desirably 4 to 15 micrometers. Further,the depth can be from 3 to 100 micrometers, and in the case where theorganic cell is a B lymphocyte, the depth is desirably 4 to 40micrometers. However, the dimensions of the microwell, as set forthabove, can be suitably determined in consideration of a desirable ratioof the diameter of the organic cell to be contained in the microwell tothe dimensions of the microwell.

The shape and size of the microwell are suitably determined inconsideration of the type of organic cell (shape, size, and the like ofthe organic cell) to be stored in the microwell so that a single organiccell will be contained per microwell.

To ensure that a single organic cell will be contained per microwell,for example, the diameter of the largest circle that can be inscribed inthe planar shape of the microwell suitably falls within a range of 0.5to 2-fold, desirably 0.8 to 1.9-fold, and preferably, 0.8 to 1.8-foldthe diameter of the organic cell to be contained in the microwell.

Further, the depth of the microwell suitably falls within a range of 0.5to 4-fold, desirably 0.8 to 1.9-fold, and preferably, 0.8 to 1.8-foldthe diameter of the organic cell to be contained in the microwell.

The number of microwells present in a single microwell array chip is notspecifically limited. However, when the organic cell is a lymphocyte andthe frequency of a given antigen-specific lymphocyte per 10⁵ cells isfrom 1 to about 500 at the high end, the number of microwells can rangefrom about 2,000 to 1,000,000 per cm², for example.

The microwell array of the present invention further comprises, on atleast a portion of the principal surface thereof around the wells, acoating layer of a substance having the ability to bind to at least aportion of a substance produced by at least a portion of the cellscontained in the wells. This is a characteristic of the microwell arrayof the present invention.

In the microwell array of the present invention, the base member can bea plate (but there is no intent to limit it to a plate), and a coatinglayer is provided on at least a portion of the principal surface aroundthe multiple wells provided on a principal surface of the base member.The microwell array of the present invention is employed to detect thepresence or absence of binding between a substance produced by at leasta portion of the cells contained in the wells and a substance in thecoating layer.

The cells contained in the wells (specimen cells) can be, for example, acell group containing immunoglobulin-producing cells orcytokine-producing cells. Further, the immunoglobulin-producing cellsand cytokine-producing cells can be natural cells, hybridomas, or cellstrains. Natural cells can be mammalian cells, such as cells collectedfrom humans, mice, or the like. Hybridomas can be created by the usualmethods described in the background art. Cell strains can be strainsinto which an expression-type cDNA library or the like has beengenetically introduced.

When the cells contained in the wells are specimen cells includingimmunoglobulin-producing cells, an antigen or an anti-immunoglobulinantibody capable of reacting to immunoglobulin in the form of asubstance produced by immunoglobulin-producing cells can be employed asthe substance having the ability to bind to at least a portion of thesubstance produced by the cells. The presence or absence of the bindingis detected using an antigen or an antibody to the immunoglobulinproduced.

When the cells contained in the wells are specimen cells includingcytokine-producing cells, an anti-cytokine antibody capable of reactingto the cytokine that is the substance produced by the cytokine-producingcells, or receptors, can be employed as the substance having the abilityto bind to at least a portion of the substance produced by the cells.The presence or absence of the binding is detected using an antibody tothe cytokine or a cytokine receptor.

Or, when the cells contained in the wells are specimen cells includingimmunoglobulin-producing cells, the substance having the ability to bindto at least a portion of the substance produced by the cells can be areceptor or cytokine receptor that is capable of reacting with theimmunoglobulin that is produced by the immunoglobulin-producing cells.In that case, the presence or absence of bonds is detected using aligand or cytokine capable of reacting with the receptor or cytokinereceptor. When the immunoglobulin produced binds to the receptor orcytokine receptor, binding to the ligand or cytokine used to detect thepresence or absence of binding is blocked, permitting detection of thepresence or absence of binding.

Examples of:

(1) substances having the ability to bind to at least a portion of thesubstance produced by the cells (binding substance);

(2) cells stored in the wells (specimen cells);

(3) substances produced by the cells (produced substances); and

(4) label substances (label substances) for identifying producedsubstances are given in Table 1 below.

The substance used to label substances for identifying the producedsubstance can be a fluorescent substance, for example. However, it isnot limited to fluorescent substances, and other label substances arepossible. The labeling with a fluorescent substance such as an antigenor antibody serving as the label substance can be conducted by the usualmethods. There are cases where the label substance binds specifically tothe produced substance, and cases where it binds specifically to thebinding substance.

TABLE 1 Produced Binding substance Cell substance Label substance Anti-B lymphocyte Immuno- Antigen immunoglobulin globulin (antibody) AntibodyAll anti- bodies such as IgG, IgM, IgA, IgE Anti- B lymphocyte Immuno-Anti- immunoglobulin globulin immunoglobulin (antibody) antibodyAntibody All anti- bodies such as IgG, IgM, IgA, IgE Anti-IgG antibody Blymphocyte IgG Antigen Anti-IgG antibody B lymphocyte IgG Anti-IgGantibody Anti-IgM antibody B lymphocyte IgM Antigen Anti-IgM antibody Blymphocyte IgM Anti-IgM antibody Anti-IgA antibody B lymphocyte IgAAntigen Anti-IgA antibody B lymphocyte IgA Anti-IgA antibody Anti-IgEantibody B lymphocyte IgE Antigen Anti-IgE antibody B lymphocyte IgEAnti-IgE antibody Anti-cytokine T lymphocyte, Cytokine Anti-cytokineantibody others antibody Anti-cytokine T lymphocyte, Cytokine (Soluble)cytokine antibody others receptor (Soluble) cytokine T lymphocyte,Cytokine Anti-cytokine receptor others antibody Antigen B lymphocyte IgGAnti-IgG antibody Antigen B lymphocyte IgM Anti-IgM antibody Antigen Blymphocyte IgA Anti-IgA antibody Antigen B lymphocyte IgE Anti-IgEantibody (Soluble) cytokine B lymphocyte Antibody Cytokine* receptor(Soluble) receptor B lymphocyte Antibody Ligand** *Detection of antibodyblocking binding of cytokine **Detection of antibody blocking binding ofligand

In forming the coating layer of a binding substance, the principalsurface of the base plate on which the coating layer is to be formed istreated with a silane coupling agent, for example, to ensure binding ofthe binding substance and the principal surface. Next, a solutioncontaining the binding substance can be applied to the surface that hasbeen treated with the silane coupling agent to form the coating layer.The amount of the coating of binding substance in the coating layer canbe suitably determined based on the type of binding substance, the typeof cell and the produced substance, and the type of label substance. Thesurface treatment to ensure binding of the binding substance to theprincipal surface is not limited to treatment with a silane couplingagent; any substance promoting binding of a binding substance comprisedof protein or the like to the surface of a base plate comprised of aninorganic material (such as a silicon material) or an organic material(such as a polymer material) can be suitably selected for use.

On the surface that is coated with the binding substance, there may becases where the binding substance may not be densely covered, withportions of uncovered surface remaining depending on the amount of thecoating. In such cases, particularly when the surface has been treatedwith a silane coupling agent as set forth above, there are cases wherethe substance produced by the cells will bind nonspecifically to thesurface of the base plate. Such nonspecific binding causes a decrease inthe precision of detection sensitivity. Accordingly, in the presentinvention, at least a portion of the principal surface not having acoating layer of a substance having the ability to bind to at least partof the substance produced by the cells that are contained in the wellsis desirably coated with a blocking agent. An example of a blockingagent is the water-soluble polymer Lipidure (registered trademark)having a structural unit in the form of2-methacryloyloxyethylphosphorylcholine (MPC) with the same structure asthe polar base of the phosphatidylcholine constituting the cellularmembrane.

[The Screening Method]

The screening method of the present invention employs the microwellarray of the present invention to screen out a target cell from a groupof cells containing specimen cells; it comprises the following steps:

(1) causing specimen cells and cell culture broth to be contained in atleast a portion of the wells of the microwell array;

(2) immersing the coating layer and the wells in culture broth andculturing the cells for some period in a state permitting the diffusionof substances contained in the culture broth from the cells into thecoating layer;

(3) after removing the culture broth, feeding a label substance bindingspecifically to the substance produced by target cells present among thespecimen cells into the coating layer; and

(4) detecting a substance that has been produced or secreted by thetarget cells and bound to the substance in the coating layer with thelabel substance to specify the target cell.

The (1) substance having the ability to bind to at least a portion of asubstance produced by a cell (binding substance); (2) cells contained inthe wells (specimen cells); (3) substance produced by the cells(produced substance); and (4) substance identifying the producedsubstance that has been labeled (label identification substance)employed in the screening method of the present invention are identicalto those described for the microwell array of the present invention, andthe items given in Table 1 are examples thereof.

Step (1)

Cells (a cell group) including specimen cells are introduced with a cellculture broth into at least a portion of the wells of a microwell array.Prior to introducing the cells, the wells of the microwell array and thearea around them are cleaned with medium; adequate removal of impuritiesthat have adhered to the surface in the course of forming the coatinglayer of a binding substance is desirable for accurate detection. Thecells are introduced with culture broth into the wells. The specimencells that are contained in the wells can be comprised of a cell groupcontaining immunoglobulin-producing cells or cytokine-producing cells.Further, the immunoglobulin-producing cells and cytokine-producing cellscan be natural cells or hybridomas. These cell groups can be obtained byknown methods.

The cells that are contained in the wells can be cells that have beenstimulated with a desired antigen in advance to induce a statepermitting substance production. For example, in the case ofimmunoglobulin-producing cells, they are desirably cells that have beenstimulated with a desired antigen in advance to induce a statepermitting the production of immunoglobulin. Similarly, in the case ofcytokine-producing cells, they are desirably cells that have beenstimulated with a desired antigen in advance to induce a statepermitting the production of cytokine. The desired antigen is notspecifically limited; various desired members of the group consisting ofproteins, sugars, lipids, nucleic acids, organic compounds, inorganiccompounds, and combinations thereof (including cells) can be suitablyselected. The culture broth can be suitably determined based on thetypes of cells that are being introduced and detected.

Step (2)

The coating layer and wells are immersed in the culture broth and thecells are cultured in a state permitting the diffusion of substancescontained in the culture broth from the wells into the coating layer.The cultured cells produce substances and the substances that areproduced are released into the culture broth, diffusing from the wellsinto the coating layer around the wells. The produced substances thatdiffuse and reach the coating layer bind to the binding substancesconstituting the coating layer. The culture conditions can be suitablydetermined based on the type of cell. The culture period can be suitablydetermined to yield a detectable quantity of produced substance bound tothe binding substance constituting the coating layer. In the coatinglayer around wells containing cells that do not produce the substance,no binding takes place between a produced substance and the bindingsubstance. An excessively long culture period results in excessivelywide diffusion of the produced substance, sometimes making it difficultto specify those wells containing cells producing the producedsubstance. The culture period is desirably suitably determined within arange permitting the ready specification of those wells containing cellsproducing the produced substance.

Step (3)

Once culturing has ended, after optionally removing the culture broth, alabel substance binding specifically to the substance produced by thetarget cells present among the specimen cells is fed into the coatinglayer. The substance produced by the target cells disperses duringculturing, binding to the binding substance constituting the coatinglayer. By feeding a label substance at this stage, the label substancebinds to the produced substance that has bound to the coating layer, orbinds to the coating layer that has not been blocked by the producedsubstance bound to the coating layer. In the former case, the labelsubstance will sometimes bind to the produced substance. In the lattercase, the label substance binds not to the produced substance, but tothe binding substance constituting the coating layer. This point isdescribed in detail further below.

Before feeding the label substance, it is desirable to remove theculture broth. When cells contained in the wells areimmunoglobulin-producing cells, for example, large amounts of antibodywill be secreted into the culture broth. When anti-immunoglobulinantibody is added, for example, it ends up binding to the antibody inthe culture broth before it binds to the antibody on the chip surface,and may preclude detection of antibody bound to the chip surface.However, there are cases where detection can be conducted withoutproblem by combining cells and binding substances even when the labelsubstance is fed into the coating layer without removing the culturebroth.

Step (4)

The substance produced by the target cell that has bound to thesubstance in the coating layer is detected by means of the labelsubstance and the target cell is specified. The label substance binds tothe produced substance that has bound to the coating layer. Accordingly,it is possible to specify the cell (target cell) producing the producedsubstance binding to the coating layer by detecting the label substance.

When the label substance is a substance binding specifically to theproduced substance, the label substance binds to the produced substancethat has diffused from the wells into the coating layer around thewells, reached the coating layer, and bound to the binding substanceconstituting the coating layer. This bound label substance is detected.Additionally, there is no binding of produced substance and bindingsubstance in the coating layer around wells in which no substance isproduced. Thus, there is no binding of the label substance, and no labelsubstance is detected.

When the label substance is a substance binding specifically to thebinding substance, the produced substance that has diffused from thewells into the coating layer around the wells, reaching the coatinglayer and binding to the binding substance constituting the coatinglayer, blocks binding of the label substance and the binding substance.Additionally, there is no binding of produced substance and bindingsubstance in the coating layer around wells in which no producedsubstance has been produced. Thus, the binding of label substance andbinding substance is not blocked, and the label substance binds. In thiscase, no label substance is detected in wells in which the producedsubstance has been produced, distinguishing them from wells in which thelabel substance is detected.

When the label substance is a fluorescent label, for example, the targetcells can be specified with a fluorescence microscope, fluorescent imagescanner, image reader, or the like.

In the embodiments described further below, chips with regularhexagonally shaped wells (lower left in FIG. 1) are primarily employed.In this case, the signal spreads in nearly concentric circle fashion(upper left in FIG. 1). However, as indicated on the lower right in FIG.1, when a diagonal slit is formed in a regular hexagonal well, theantibody or the like that is produced also spreads in the direction ofthe slit, yielding a shape such as the Milky Way system shown in theupper right of FIG. 1. In this manner, the signal from the labelsubstance obtained via the product varies in shape based on the shape ofthe well.

When the Label Substance is a Substance Binding Specifically to theProduced Substance (1)

When the specimen cells contained in the wells includeimmunoglobulin-producing cells, culturing the cells results in theproduction of immunoglobulin. By using a coating layer with ananti-immunoglobulin antibody or antigen as the binding substance, theimmunoglobulin that is produced binds to the coating layer, and theimmunoglobulin that has been produced and has bound to the coating layercan be detected using an antibody to the immunoglobulin or an antigen.Around wells containing cells that do not produce immunoglobulin, thatis, around wells containing cells that do not produce immunoglobulinwhen stimulated with the antigen that has been provided, there is noimmunoglobulin that has diffused from the wells. These wells can bedistinguished from wells containing cells that produce immunoglobulin.In this manner, it is possible to specify target cells in the form ofantigen-specific immunoglobulin-producing cells.

A more specific description will be given using FIG. 2.

(A) Antibody-secreting cells are poured into the individual wells of amicrowell array chip made of silicon to the surface of whichanti-immunoglobulin (Ig) antibody has been bound.

(B) The cells are cultured, causing them to secrete antibody. Theantibody that is secreted by the cells binds to the anti-1 g antibodyaround the wells.

(C) When fluorescence-labeled antigen is added, the antigen binds to theantigen-specific antibody that has been trapped around the wells.

(D) Observation by fluorescence microscopy, scanning, or the likereveals that the antigen-specific antibody has spread into a donut shapearound the wells.

When the Label Substance is a Substance Binding Specifically to theProduced Substance (2)

When the specimen cells contained in the wells includecytokine-producing cells, culturing the cells results in the productionof cytokine. By using a coating layer with an anti-cytokine antibody orcytokine receptor as the binding substance, the cytokine that isproduced binds to the coating layer, and the cytokine that has beenproduced and bound to the coating layer can be detected using anantibody to the cytokine or a cytokine receptor. Around wells containingcells that do not produce cytokine, that is, wells containing cells thatdo not produce cytokine when stimulated with the antigen that has beenprovided, no cytokine diffuses from the wells. These wells can bedistinguished from wells containing cells that produce cytokine. In thismanner, it is possible to specify target cells in the form ofcytokine-producing cells.

When the Label Substance is a Substance Binding Specifically to theBinding Substance

When the specimen cells contained in the wells includeimmunoglobulin-producing cells, culturing the cells results in theproduction of immunoglobulin. By using a coating layer with a cytokinereceptor or receptor as the binding substance, a portion of theimmunoglobulin that is produced binds to the coating layer, and theportion of the immunoglobulin that has been produced and has bound tothe coating layer blocks binding of the cytokine receptor or receptor tothe label substance in the form of a cytokine or a ligand. The labelsubstance does not bind around wells containing cells producingimmunoglobulin. Around wells containing cells that do not produceimmunoglobulin, that is, wells containing cells that do not produceimmunoglobulin when excited with the antigen that has been provided, noimmunoglobulin disperses from the wells, and the binding of cytokinereceptor or receptor and cytokine or ligand is not blocked. Around wellscontaining cells that produce immunoglobulin that is not bound by acytokine receptor or receptor, and around wells containing cellsproducing immunoglobulin that is bound by a cytokine receptor orreceptor, but binding between cytokine receptor or receptor and cytokineor ligand is not blocked, the immunoglobulin diffusing from the wellsbinds, but the binding of cytokine receptor or receptor to the cytokineor ligand is not blocked. Such wells can be distinguished from wellscontaining cells producing immunoglobulin. Target cells in the form ofantigen-specific immunoglobulin-producing cells can thus be specified.

The above method will be described based on FIG. 3.

(A) A receptor is coated on the chip surface.

(B) Antibody-secreting cells are added to the microwells and cultured.The antibody that is secreted diffuses. The receptor-specific antibodybinds to the receptor around the wells.

(C) When the antibody that has bound to the receptor binds to aligand-binding site, fluorescence-labeled ligand cannot bind to thereceptor.

(D) When the antibody that has bound to the receptor binds to somethingother than a ligand-binding site, fluorescence-labeled ligand is able tobind to the receptor.

[The Method for Producing Target Cells]

The present invention includes a method for producing target cellscomprising recovering from the wells target cells that have beenspecified by the above-described screening method of the presentinvention. The target cells can be specific immunoglobulin-producingcells or specific cytokine-producing cells.

The present invention permits the preparation of an antigen-specificantibody protein by recovering an antigen-specific immunoglobulin genefrom a recovered target cell, expressing antibody cDNA and preparing anantigen-specific antibody protein. Further, the mRNA of a T cellreceptor gene expressed by a cell that has produced a cytokine can berecovered, and the T cell receptor cDNA can be recovered and introducedinto another cell to express a T cell receptor protein.

EMBODIMENTS

The present invention is described in greater detail below based onembodiments.

An example of the preparation of a microwell array chip will bedescribed.

Preparation Example 1

A preparation example employing a silicon base plate will be described.FIG. 4-1 is an example of a microwell array chip based on PreparationExample 1. Examples of the preparation steps are shown in FIG. 4-2.

(1) An oxide film 1-a is formed on a silicon base plate 1-b.

(2) A photoresist 1-c, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied on the base plate and an exposure device is used to transfera pattern.

(3) Oxide film 1-a that is exposed through photoresist 1-c is etchedwith buffered hydrofluoric acid.

(4) Microwells 1-d that are 10 to 20 microns in depth are formed by dryetching or wet etching. Photoresist 1-c is suitably removed based on theetching method.

(5) The surface is treated with primer so that antibodies will binduniformly. For example, a silylation treatment is used to form silylgroups 1-e on the surface and inner walls of the wells. There arevarious materials and methods that can be employed in this treatment;they are not limited by the treatment employed in the presentpreparation example.

(6) The detection of antibody-secreting cells is conducted based on thepresent invention.

Preparation Example 2

To more clearly monitor the antibodies that are secreted by cells, it ispossible to provide partitions around wells. FIG. 5-1 shows the externalappearance of such a partition and FIG. 5-2 shows a preparation example.

(1) An oxide film 2-a is formed on a silicon base plate 2-b.

(2) A photoresist 2-c, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied on the base plate and an exposure device is used to transfera pattern.

(3) Oxide film 2-a that is exposed through photoresist 2-c is etchedwith buffered hydrofluoric acid.

(4) Photoresist 2-c is removed and indentations 2-d that are 1 to 5microns in depth are fabricated by etching.

(5) A photoresist 2-e, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied again on the base plate and an exposure device is used totransfer a pattern. When the indentations are deep, a negative resistsuch as OMR-85 from Tokyo Ohka Kogyo Co., Ltd. can be employed.

(6) Wells 2-f that are 10 to 20 microns in depth are formed with a dryetcher.

(7) The surface is treated with primer so that antibodies will binduniformly. For example, a silylation treatment is used to form silylgroups 2-g on the surface and inner walls of the wells. There arevarious materials and methods that can be employed in this treatment;they are not limited by the treatment employed in the presentpreparation example.

(8) The detection of antibody-secreting cells is conducted based on thepresent invention. In the present example, since walls are formed aroundthe wells to inhibit the diffusion of antibodies, muddling due to thediffusion of antibodies is inhibited, facilitating image identification.

FIG. 5-3 shows the result of a trial run of the present preparationexample.

Preparation Example 3

By increasing the surface area around the wells, it is possible to moreaccurately observe antibodies that are secreted. The surface area can beincreased by forming a porous silicon structure or an uneven structure,for example. Japanese Unexamined Patent Publication (KOKAI) Heisei No.6-21509, for example, describes a method for preparing porous silicon.An uneven structure can be prepared by etching, vapor deposition, or thelike. FIG. 6-1 shows the external appearance thereof, and FIG. 6-2 showsa preparation example thereof.

(1) An oxide film 3-a is formed on silicon base plate 3-b.

(2) A photoresist 3-c, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied on the base plate and an exposure device is used to transfera pattern.

(3) Oxide film 3-a that is exposed through photoresist 3-c is etchedwith buffered hydrofluoric acid.

(4) To increase the surface area of openings in exposed portions, aporous structure 3-d, for example, can be fabricated. The porousstructure is formed by anode chemical conversion treatment or the like.A nanostructure can also be fabricated on the surface with a mixture ofhydrofluoric acid and nitric acid.

(5) A photoresist 3-e, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied again on the base plate and an exposure device is used totransfer a pattern. Dry etching is used to form wells 3-f that are 10 to20 microns in depth.

(6) The surface is treated with primer so that antibodies will binduniformly. For example, a silylation treatment is used to form silylgroups 3-g on the surface and inner walls of the wells. There arevarious materials and methods that can be employed in this treatment;they are not limited by the treatment employed in the presentpreparation example.

(7) The detection of antibody-secreting cells is conducted based on thepresent invention. In the present example, since the surface area isincreased around the wells and since the density of fluorescence-labeledbonds is increased, fluorescence image identification is facilitated.

Preparation Example 4

By causing antibody to bind in localized fashion only around the wells,it is possible to observe antibody secretion more clearly. In thepresent method, a substance serving as a means of causing antibody tobind only around the wells can be achieved by termination using apattern or the like. The external appearance thereof is shown in FIG.7-1, and a preparation example is shown in FIG. 7-2. An oxide film 4-ais formed on a silicon substrate 4-b.

(1) A photoresist 4-c, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.is applied on the base plate and an exposure device is used to transfera pattern.

(2) Oxide film 4-a that is exposed through photoresist 4-c is etchedwith buffered hydrofluoric acid.

(3) The surface is treated with primer so that antibodies will binduniformly. For example, the surface is terminated with a silane couplingagent in the form of hexamethyldisilazane or the like. There are variousmaterials and methods that can be employed in this treatment; they arenot limited by the treatment employed in the present preparationexample.

(4) A photoresist 4-e, such as OFPR-800 from Tokyo Ohka Kogyo Co., Ltd.,is applied thereover.

(5) An antibody binding pattern is formed on photoresist 4-e with anexposure device. The dimension of the antibody binding pattern, which isgreater than the wells, is from 1 micron to ½ the spacing of adjacentwells.

(6) Photoresist 4-e is processed into a well pattern with an exposuredevice.

(7) Dry etching is used to form wells 4-f that are 10 to 20 microns indepth in the well pattern. In the course of forming wells, substance 4-dthat binds to antibody that is unprotected by photoresist 4-e issimultaneously removed. It can also be removed with oxygen plasma or thelike.

(8) Photoresist 4-e is removed with an organic solvent such as acetone.Since the substance binding to antibodies is present only around thewells, the antibody binding range can be localized.

(9) The above steps can be simplified by employing a photosensitivesilane coupling agent. Specifically, after conducting processingaccording to Preparation Example 1, the photosensitive silane couplingagent is applied and formed into a desired pattern by exposure to lightto achieve the same results as in the present preparation example.

Fluorescence-labeled antibody was sown onto a microwell array chipproduced according to the present preparation example. The binding stateobserved is shown in FIG. 7-3. Antibody binding was observed only inportions around the wells. Further, the present preparation example ischaracterized in that the antibody binding surface was formed onlyaround the wells; there are various methods of achieving this. It isalso possible to employ the dix series or the like made by KishimotoSangyo Co., Ltd. It forms amino groups on the surface ofpolyparaxylylene resin. The same binding surface can be realized as inthe present preparation example. The same effect can also be achieved byforming a film having an effect that is the reverse of that of theprimer for antibody binding on portions other than the antibody bindingsurface as needed. An example is Biosurfine, made by Toyo Gosei Co.,Ltd.

The Surface Treatment Method (FIG. 8)

A surface treatment is conducted by the following method to causeanti-Ig antibody to bind uniformly.

1. Grime, such as oil, on the surface of base plate 5-a is removed. Thegrime is removed, for example, by cleaning with an organic solvent,acid, and alkali, or by dry cleaning with oxygen plasma or the like. Thebase plate material can be suitably selected based on the grime.

2. To increase the surface binding density of primer binding the baseplate and an antibody, for example, it is also possible to form hydroxylgroups 5-b that substitute onto the primer on the surface of the baseplate. To form hydroxyl groups on the surface of the base plate andremove microparticles from the surface, washing with about 1 percentammonia water is conducted. Thorough rinsing with water is thenconducted. In the case of silicon, it suffices to substitute this forSC-1 cleaning in RCA cleaning. However, 2. can be omitted if theformation of a surface suited to adequate primer binding can be achievedin 1.

3. Primer 5-c is bound to the surface. It is immersed in a silanecoupling agent or the like, and adequately substituted with hydroxylgroups on the surface. The primer is, for example, hexamethyldisilazane,which renders the surface hydrophobic. There are various primermaterials and methods that can be employed in this treatment; they arenot limited to the surface treatment agent employed in the presentpreparation example.

The primer is chiefly characterized by being a silane coupling agent ora silylating agent, and by binding organic material to inorganicmaterial. It is employed as a surface-modifying agent, and produces ahydrophobic surface. In semiconductors, materials typified byhexamethyldisilazane are primarily employed. The primer is needed to besuitably selected based on the base plate material. Examples are thesilane coupling agents and silylating agents sold by Shin-Etsu Silicone.

The primer can be coated by a variety of methods. Dipping, spin coating,gas diffusion, N₂ bubbling, and the like are all possible. It isnecessary to select the method best suited to the material. For example,in the case of gas diffusion, implementation is possible by exposure toan atmosphere of vaporized primer in a tightly sealed container for 5 to10 minutes.

The microwell array chip of the present preparation example is notlimited to silicon, and can be formed from resin, metal, glass, or thelike. It need not be in the form of a single material, but can be in theform of a film formed on a base plate.

Examples of resins are acrylic, polypropylene, polyethylene, polyvinylchloride, ABS, polyurethane, epoxy resin, thermosetting resins,photocuring resins, and photo-soluble resins. These resins can be moldedby injection, compression, thermosetting, photocuring, dry etching, andthe like. Molding can also be conducted by forming a resin film on aglass, silicon, or metal base plate. The surface of a resin that hasbeen molded into the shape of microwells can be treated with primer inthe same manner as silicon to bind antibody. For example, this can berealized by the surface treatment method described in the presentinvention. By using a photosensitive primer, it is possible to form anantibody binding pattern in the same manner as on silicon. Here, theprimer employed is suitably selected while also considering its bindingproperty to resin materials. If the resin itself functions to coupleantibody to the surface thereof, and is capable of binding antibody, aneven greater reduction in cost can be achieved.

Examples of metals are aluminum, aluminum alloys, copper alloys, gold,and stainless steel. These metals can be molded by molding in metalmolds, etching, and the like. Molding can also be conducted by forming ametal film on a glass, silicon, or metal base plate. A surface of metalthat has been molded into the shape of microwells can be treated withprimer in the same manner as silicon to bind antibody. For example, thiscan be realized by the surface treatment method described in the presentinvention. Here, the primer employed is suitably selected while alsoconsidering its binding property to metal materials.

Microwells can be formed in glass by etching. Further, a film of resin,silicon, or metal can be formed on the surface to form microwells. Thesurface of a metal that has been molded into the shape of microwells canbe treated with primer in the same manner as silicon to bind antibody.For example, this can be realized by the surface treatment methoddescribed in the present invention. Here, the primer employed issuitably selected while also considering its binding property to glassand surface-film forming materials.

In the embodiments below, silicon microwell array chips fabricatedaccording to Preparation Example 1 were employed. However, the surfaceshape of the wells of the chips employed in the embodiments washexagonal (round in Preparation Example 1).

Embodiment 1 Experimental Method Protocol I

1. [Applying a coating on an anti-human IgG chip] An 80 μL quantity ofanti-human IgG that had been diluted with PBS to 10 μg/mL was added to asilicon microwell array chip that had been treated with a silanecoupling agent, the chip was placed in a moisture-conserving box (FIG.9) to maintain the chip humidity so that the solution on the chip didnot dry out, and the chip was left standing for one hour at roomtemperature (15 to 25° C.) to cause the anti-IgG antibody to bind to thesurface of the silicon chip. In this case, in contrast to step 2 below,no degassing by pressure reduction was conducted. The antibody did notenter the wells, but was distributed around the wells. (Subsequently,while being incubated, the chip was constantly kept in themoisture-conserving box to prevent it from drying out.)

2. [Blocking] The antibody solution was removed from the chip, 100 μL ofPBS was added to the chip and then removed in a cleaning operation thatwas repeated three times. Subsequently, 100 μL of PBS containing 0.2percent (v/v) Lipidure (NOF Corporation, BL-B03 Lipidure (5 weightpercent)) was added to the chip, and a vacuum pump was used to generatea vacuum to completely remove bubbles in the microwells, therebycovering the surface of the chip and filling the interior of the wellswith Lipidure solution. The chip was left standing for 15 minutes atroom temperature (15 to 25° C.) to conduct blocking.

3. [Adding cells] The chip was washed three times with 100 μL of RPMI1640 medium containing 10 percent FCS. X63/116 cells (secreting humanIgG in response to hepatitis B virus surface antigen (HBs antigen)) orHyHEL10 110TC cells (secreting mouse/human chimera antibody in responseto chicken egg lysozyme (HEL)) that had been washed twice with RPMI 1640medium containing 10 percent FCS were added to the chip and the chip wasleft standing at room temperature for about 10 minutes so that the cellsentered the wells.

4. [Culturing the cells] Cells that had not entered the wells wereremoved by washing several times with RPMI 1640 medium containing 10percent FCS (until all of the surplus cells had been removed). An 80 μLquantity of RPMI 1640 medium containing 10 percent FCS was added ontothe chip, the chip was placed in a CO₂ incubator (37° C., 5 percentCO₂), and the cells were cultured on the chip for 2 to 3 hours.

5. [Binding biotin-labeled antigen] While taking care to prevent thecells from exiting the wells, the buffer was removed from the chipsurface, about 100 μL of PBS was added to the surface of the chip andthen removed, and this operation was repeated several times to clean thechip. Subsequently, an 80 mL quantity of 1 mg/mL biotin-labeled HEL orbiotin-labeled HBs antigen was added to the chip and the chip was leftstanding for 30 minutes at room temperature.

6. After washing the chip with PBS in the same manner as in 5, 80 μL ofCy3-labeled streptavidin (Sigma, S-6402) comprised of a 1,000-folddilution of the original solution was added to the chip, and the chipwas similarly left standing for 30 minutes at room temperature.

7. After washing the chip with PBS in the same manner as in 5, PBS wasadded to the chip, the fluorescence of the Cy3 was observed byfluorescence microscopy, and the positions of wells containing cellssecreting antigen-specific antibody were determined based on thespreading of Cy3 fluorescence in the form of donuts around the wells(FIG. 10).

8. An 80 μL quantity of 1 μg/mL Oregon green was added to the chip andthe chip was left standing for 3 minutes at room temperature to labelthe cells with Oregon green. After washing the chip with PBS in the samemanner as in 5, fresh PBS was added to the chip. While observing thecells using the fluorescence of the Oregon green as an indicator, targetcells that were secreting antigen-specific antibody were recovered witha micromanipulator.

Identification of Antigen-Specific Antibody-Secreting Cells on aMicrowell Array Chip by the Method of the Present Invention (FLISPOTMethod)

HyHEL10 110TC cells, X63/116 cells, and 110TC cells (negative control,did not secrete antibody) were cultured on silicon microwell array chipscoated with anti-IgG antibody. The secretion of antigen-specificantibody was detected with biotin-labeled HEL or biotin-labeled HBsantigen and Cy3-labeled streptavidin (FIG. 11A). As indicated in FIG.11A, the antibody that had been secreted was detected in the form ofdonuts around the wells. On the chip to which 110TC that did not secreteantibody had been added, no signal was detected. Oregon green was thenused to stain the cells and the cells were confirmed (FIG. 11B).

The staining was antigen-specific. On the chip to which HyHEL10 110TCcells had been added, a signal was detected with biotin-labeled HEL, butno signal was detected with biotin-labeled HBs antigen. Conversely, onthe chip to which X63/116 cells had been added, no signal was detectedwith biotin-labeled HEL, but a signal was detected with biotin-labeledHBs antigen (the data are not given).

Identification of Antigen-Specific Antibody-Secreting Hybridomas by theFLISPOT Method (Applying Protocol I)

Spleen cells were prepared from BALB/c mice that had been immunized withHEL protein. These cells were fused by the usual method employingpolyethylene glycol with X63.Ag8.653 myeloma cells to create hybridomas.The hybridomas were selected with HAT selection medium and then added toa microwell array chip that had been coated with anti-mouse IgGantibody. The cells that did not enter the wells were removed bywashing, after which the cells were cultured on the chip for 1 hour 30minutes. Hybridomas secreting antibody to HEL were detected usingbiotin-labeled HEL and PE-labeled streptavidin (FIG. 11A). Oregon greenwas then added to determine the position of the cells (FIG. 11B). (FIG.11C) A combination of A and B.

Embodiment 2 Experimental Method Protocol II

Preparation of Mouse CD138 Positive Cells (Antibody-Secreting Cells)

1. Mouse spleen cells were prepared.

2. Anti-mouse CD138 antibody was added to a 100 μL cell suspension in aquantity of <1 μg per 10⁶ cells and the cells were incubated for 15minutes at 4° C.

3. The cells were washed twice with 10 mL of PBS and then suspended in100 μL of PBS. To the cell suspension was then added anti-ratκ(kappa)-chain antibody-bound microbeads in a quantity of <1 μg per 10⁶cells and the cells were incubated for 15 minutes at 4° C.

4. The cells were washed twice with 10 mL of PBS and then suspended in1,000 μL of PBS. CD138 positive cells were recovered with an AutoMACS.

Preparation of Human CD138 Positive Cells

1. Lymphocytes were separated from human peripheral blood by anestablished method (the specific gravity centrifugation method employingFicoll).

2. Fc receptor blocking reagent (Miltenyl Biotec Co., Ltd.) was added ina quantity of 20 μL per 10⁷ cells, followed by anti-CD138 antibody-boundmicrobeads in a quantity of <1 μg per 10⁶ cells and the cells wereincubated for 15 minutes at 4° C.

3. The cells were washed twice with 10 mL of PBS and suspended in 1,000μL of PBS. The CD138 positive cells were then recovered with anAutoMACS.

Experimental Method Protocol II, Continued

1. In the tests below, the same measures were adopted to preventingdrying out as in protocol I in the course of incubation.

2. To a silicon microwell array chip that had been treated with a silanecoupling agent was added 80 μL of PBS-diluted 10 μg/mL donkey-derivedanti-goat IgG antibody. The chip was placed in a moisture-conserving box(FIG. 1) so that the solution on the chip did not dry out, and leftstanding for 1 hour at room temperature (15 to 25° C.) to cause thedonkey-derived anti-goat IgG antibody to bind to the surface of thesilicon chip.

3. The antibody solution was removed from the chip, the chip was washedthree times with 100 μL of PBS, 100 μL of PBS containing 0.2 percent(v/v) Lipidure (NOF Corporation, BL-B03 Lipidure (5 weight percent)) wasadded to the chip, and a vacuum was generated to remove bubbles in themicrowells, thereby covering the surface of the chip and filling theinterior of the wells with Lipidure solution. The chip was left standingfor 15 minutes at room temperature to conduct blocking.

4. The chip was washed three times with 100 μL of RPMI 1640 mediumcontaining 10 percent FCS. CD138 positive antibody-secreting cells (1 to2×10⁶ cells suspended in 30 μL of PBS) that had been washed once with 10mL of PBS were added to the chip and the chip was left standing forabout 10 minutes at room temperature to allow the cells to enter thewells.

5. Cells that had not entered the wells were removed by washing withRPMI 1640 medium containing 10 percent FCS. An 80 μL quantity of 10μg/mL goat-derived anti-human (or mouse) IgG was then added to the chipand the chip was left standing for 30 minutes at room temperature tocause the antibody to bind to the donkey-derived goat IgG antibody onthe chip surface.

6. The chip was washed with RPMI 1640 medium containing 10 percent FCSwhile taking care to prevent the cells from exiting the wells. An 80 μLquantity of RPMI 1640 medium containing 10 percent FCS was then added tothe chip and the cells were cultured for 3 hours in a CO₂ incubator (37°C., 5 percent CO₂).

7. Subsequent steps in the form of step 5 and beyond in protocol I werethen conducted.

Detection of HEL-Specific Antibody-Secreting Cells Among Mouse SpleenCells Immunized with HEL by the Method of the Present Invention (FLISPOTMethod)

CD138 positive cells were prepared from the spleen cells of BALB/c micethat had been immunized with chicken egg lysozyme (HEL). The cells wereadded to a silicon microwell array chip to which donkey-derivedanti-goat IgG antibody had been bound according to protocol II.Subsequently, goat-derived anti-mouse IgG antibody was bound to the chipaccording to the protocol and the cells were cultured for about 3 hours.Subsequently, biotin-labeled HEL and Cy3-labeled streptavidin were addedand the cells that were secreting HEL-specific IgG antibody weredetected by fluorescence microscopy (FIG. 12). Subsequently, the cellswere recovered with a micromanipulator and the antibody gene wasamplified. Of the seven IgG's prepared, six bound to HEL (the data arenot given).

Detection of HBs-Specific Antibody-Secreting Cells Among HumanPeripheral Blood Lymphocytes by the Method of the Present Invention(FLISPOT Method)

CD138 positive cells were prepared by protocol II from healthy humanperipheral blood that had been boosted with HBs antigen vaccine. TheCD138 positive cells were added to a silicon microwell array chip towhich donkey-derived anti-goat IgG antibody had been bound by protocolII. Subsequently, goat-derived anti-human IgG antibody was bound to thechip according to the protocol and the cells were cultured for about 3hours. Biotin-labeled HBs antigen and Cy3-labeled streptavidin were thenadded and the cells that were secreting HBs antigen-specific IgGantibody were detected by fluorescence microscopy (FIG. 13).

Volunteers from whom informed consent had been obtained were inoculatedwith HBs vaccine, 100 mL of blood was drawn on day 7, CD138 positivecells were selected from peripheral blood lymphocytes obtained from thisblood, and these cells were sown on a plate that had been coated withanti-human IgG. The cells were cultured, after which HBs-specificantibody-secreting cells were detected using biotin-labeled HBs antigenand Cy3-labeled streptavidin. Cells were recovered from a total of 24wells. HBs-specific antibody-secreting cells were similarly detectedfrom peripheral blood lymphocytes obtained by drawing 100 mL of blood onday 8 following HBs vaccination, and cells were recovered from 57 wells.From these cells, 53 pairs of antibody H chain and L chain cDNA wereprepared. The cDNA was combined into an expression vector, the pairs ofH chain and L chain were introduced into the genome of 293T cells (acell strain derived from fetal human kidney), and the culturesupernatant was recovered. Whether or not the antibody that had beensecreted into the supernatant bound to HBs antigen was analyzed byELISA. As a result, 41 antibody proteins were produced, of which 36 wereHBs antigen-specific.

Embodiment 3 Experimental Method Protocol III

1. An 80 μL quantity of antigen (HEL protein) that had been diluted withPBS to 10 μg/mL was added to a silicon microwell array chip that hadbeen treated with a silane coupling agent. The chip was placed in amoisture-conserving box (FIG. 9) so that the solution on the chip wouldnot dry out. The chip was left standing for 1 hour at room temperature(15 to 25° C.) to cause the HEL protein to bind to the surface of thesilicon chip. (Each time the chip was subsequently incubated, it wasplaced in this box to prevent drying out.)

2. The antigen solution was removed from the chip and the chip waswashed three times with 100 μL of PBS. A 100 μL quantity of PBScontaining 0.2 percent (v/v) Lipidure (NOF Corporation, BL-B03 Lipidure(5 weight percent)) was added to the chip, and a vacuum was generated toremove bubbles in the microwells, thereby covering the surface of thechip and filling the interior of the wells with Lipidure solution. Thechip was left standing for 15 minutes at room temperature to conductblocking.

3. CD138 positive cells were prepared according to protocol II frommouse spleen cells inoculated with HEL.

4. The chip was washed with RPMI 1640 medium containing 10 percent FCS,after which CD138 positive cells (1 to 2×10⁶ cells suspended in 30 μL ofPBS) that had been washed once with 10 mL of PBS were added to the chip.The chip was then left standing for about 10 minutes to allow the cellsto enter the wells.

5. Cells that had not entered wells were removed by washing with RPMI1640 medium containing 10 percent FCS, after which 80 μL of RPMI 1640medium containing 10 percent FCS was added to the chip. The chip wasthen placed in a CO₂ incubator (37° C., 5 percent CO₂) and the cellswere cultured for 3 hours on the chip.

6. Taking care not to cause the cells to exit the wells, the buffer onthe surface of the chip was removed, 100 μL of PBS was added to thesurface of the chip, and this operation was repeated several times toclean the chip. Subsequently, 80 μL of 1 μg/mL Cy3-labeled anti-mouseIgG was added to the chip and the chip was left standing for 30 minutesat room temperature.

7. A chip identical to that in 6 was washed with PBS, PBS was added tothe chip, and the fluorescence of the Cy3 was observed by fluorescencemicroscopy to determine the positions of wells containing cells thatwere secreting antigen-specific antibody using the spreading of Cy3fluorescence in the shape of donuts around the wells as an indicator(FIG. 14).

8. An 80 μL quantity of Oregon green was added to the chip and the chipwas left standing at room temperature for 3 minutes to label the cellswith Oregon green. A chip identical to that in 6 above was washed withPBS, fresh PBS was added to the chip, and while observing the cells withthe fluorescence of the Oregon green as indicator, the target cells thatwere secreting antigen-specific antibody were recovered with amicromanipulator.

Embodiment 4 Experimental Method Application of Protocol IV

The Use of Antigen Labeled with Enzyme or Antibody Labeled with EnzymeInstead of Fluorescence-Labeled Antigen or Fluorescence-Labeled Antibody

Differences

1. After employing the action of an enzyme-labeled antigen orenzyme-labeled antibody, a buffer to which the substrate of the enzymehad been added was added to the chip and incubation was conducted atroom temperature.

2. The product resulting from conversion of the substrate by the enzymeprecipitated in ring form around the well. This was observed by opticalmicroscopy, permitting the detection of antibody-secreting cells.

Enzyme: alkaline phosphatase; Substrate: BCIP/NBT

Detection is also possible using an antigen or an antibody to which aquantum dot has been bound instead of a fluorescent pigment.

Detection of Cytokine-Producing Cells

1. Instead of causing anti-IgG antibody to bind to the chip,anti-cytokine antibody is caused to bind to the chip, T cells or thelike that secrete cytokine are sown on the chip, and the production ofcytokine is induced.

2. The cytokine that is secreted binds to the anti-cytokine antibodyaround the wells.

3. Next, when fluorescence or enzyme-labeled cytokine antibody is added,the rings of cytokine that binds around the wells can be detected andthe cytokine-secreting T cells or the like can be detected.

Immuno Spot Assay Assay on Chip (ISAAC)

The Detection of Cytokine-Secreting Cells

Method

1. Lymphocytes were prepared from human peripheral blood and stimulatedovernight in a CO₂ incubator (37° C., 5 percent CO₂) in the presence of10 ng/mL phorbol myristate acetate (PMA) and 1 muM ionomycine.

2. The lymphocytes were recovered and washed, after which the cells wereadded to a microwell array chip that had been coated with anti-human IFNγ(gamma)-antibody. The chip was placed in a moisture-conserving box(FIG. 9) to prevent drying out, after which the cells were cultured for5 hours in a CO₂ incubator (37° C., 5 percent CO₂).

3. The chip was washed with PBS-Tween20, biotin-labeled anti-human IFN γ(gamma)-antibody was added to the chip, and the chip was incubated for30 minutes at room temperature in a moisture-maintaining box.

4. The chip was washed with PBS-Tween20, Cy3-labeled streptoavidin wasadded, and the chip was incubated for 30 minutes at room temperature inthe moisture-maintaining box.

5. The chip was washed with PBS-Tween20 and the signal of the cytokinesecreted was observed by fluorescence microscopy (2 second exposure).

The results are given in FIG. 15.

Embodiment 5 Obtaining Blocking Antibody

1. The same measures to prevent drying out were adopted as in protocol 1in the course of incubating the chips in the following tests.

2. An 80 μL quantity of 10 μg/mL of TRAIL-R1/Fc diluted with PBS wasadded to a silicon microwell array chip that had been treated with asilane coupling agent, and the chip was placed in a moisture-conservingbox (FIG. 9) so that the solution on the chip did not dry out. The chipwas left standing for 1 hour at room temperature (15 to 25° C.) and theTRAIL-R1/Fc was bound to the surface of the silicon chip.

3. The antibody solution was removed from the chip and the chip waswashed three times with 100 μL of PBS. A 100 μL quantity of PBScontaining 0.2 percent (v/v) Lipidure (NOF Corporation, BL-B03 Lipidure(5 weight percent)) was added to the chip, and a vacuum was generated toremove bubbles in the microwells, thereby covering the surface of thechip and filling the interior of the wells with Lipidure solution. Thechip was left standing for 15 minutes at room temperature to conductblocking.

4. The chip was washed three times with 100 μL of RPMI 1640 mediumcontaining 10 percent FCS. CD138 positive antibody-secreting cells thathad been washed once with 10 mL and washed twice with 1,000 μL of RPMI1640 medium containing 10 percent FCS (1 to 2×10⁶ cells suspended in 30μL of RPMI 1640 medium containing 10 percent FCS) were added to thechip, after which the chip was left standing for about 10 minutes atroom temperature to allow the cells to enter the wells.

5. Taking care not to cause the cells to exit the wells, the chip waswashed with RPMI 1640 medium containing 10 percent FCS, after whichanother 80 μL of RPMI 1640 medium containing 10 percent FCS was added tothe chip. The cells were then cultured for 3 hours in a CO₂ incubator(37° C., 5 percent CO₂).

6. Taking care not to cause the cells to exit the wells, the buffer wasremoved from the surface of the chip, the operation of adding about 100μL of PBS to the chip surface and removing it was repeated several timesto clean the chip, 80 μL of 1 μg/mL biotin-labeled TRAIL was added tothe chip, and the chip was left standing for 30 minutes at roomtemperature.

7. After washing the chip with PBS in the same manner as in 6, 80 μL ofCy3-labeled streptavidin (Sigma, S-6402) comprising an original solutiondiluted 1,000-fold, was added to the chip, which was then similarly leftstanding for 30 minutes at room temperature.

8. After washing the chip with PBS in the same manner as in 6, PBS wasadded to the chip, and the fluorescence of the Cy3 was observed byfluorescence microscopy. The positions of wells containing cells thatwere secreting antibody that was being blocked between the TRAIL-R1/Fcand its ligand, TRAIL, were determined using an indicator in the form ofblack donut shapes where the fluorescence of Cy3 had not bound aroundwells on the surface of the chip that was bright red with Cy3fluorescence.

9. An 80 μL quantity of 1 μg/mL Oregon green was added to the chip andthe chip was left standing at room temperature for 3 minutes to labelthe cells with Oregon green. After washing the chip with PBS in the samemanner as in 6, fresh PBS was added to the chip. While observing thecells with the fluorescence of Oregon green as an indicator, the targetcells that were secreting antigen-specific antibody were recovered witha micromanipulator.

Embodiment 6 Application of Functional Antibody to Screening

Receptor protein was coated on the surface of a chip, and CD138 cellsprepared from the spleen cells of mice that had been immunized withreceptor protein were sown on the chip. The cells were cultured, afterwhich biotin-labeled ligand and Cy3-labeled streptavidin were added.Biotin-labeled ligand and Cy3-labeled streptavidin was bound over nearlythe entire surface of the chip. However, the binding of biotin-labeledligand and Cy3-labeled streptavidin was blocked around the wells ofcells producing a functional antibody that blocked the binding ofreceptor ligand, so these areas were dark. The results are shown in FIG.16. Left: Detection results for biotin-labeled ligand/Cy3-labeledstreptavidin. Middle: The cells were labeled with Oregon green andobserved. Left: The left and middle figures superposed.

INDUSTRIAL APPLICABILITY

The present invention is useful in technical fields relating to methodsof screening immune cells, such as specific immunoglobulin-producingcells and specific cytokine-producing cells. Examples are the fields ofdiagnosis and immunology-related pharmaceuticals, including antibodypharmaceuticals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the relation between the form of the signalfrom a label substance and the shape of the well.

FIG. 2 is a descriptive drawing of the method of the present inventionin an example employing antibody-secreting cells.

FIG. 3 is a descriptive drawing of the method of the present inventionin case (1), in which the label substance binds specifically to aproduced substance.

FIG. 4-1 is a descriptive drawing of Preparation Example 1.

FIG. 4-2 is a descriptive drawing of Preparation Example 1.

FIG. 5-1 is a descriptive drawing of Preparation Example 2.

FIG. 5-2 is a descriptive drawing of Preparation Example 2.

FIG. 5-3 is a descriptive drawing of Preparation Example 2.

FIG. 6-1 is a descriptive drawing of Preparation Example 3.

FIG. 6-2 is a descriptive drawing of Preparation Example 3.

FIG. 7-1 is a descriptive drawing of Preparation Example 4.

FIG. 7-2 is a descriptive drawing of Preparation Example 4.

FIG. 7-3 is a descriptive drawing of Preparation Example 4.

FIG. 8 is a descriptive drawing of a surface treatment method.

FIG. 9 is a descriptive drawing of a moisture-conserving box thatmaintains the humidity of the chip.

FIG. 10 shows the results of the determination of the positions of wellscontaining cells secreting antigen-specific antibodies in Embodiment 1.

FIG. 11 shows the results of identification on a microwell array chip ofantigen-specific antibody-secreting cells employed in the method(FLISPOT) of the present invention in Embodiment 1.

FIG. 12 shows the results of detection of HEL-specificantibody-secreting cells among mouse spleen cells immunized with HEL bythe method (FLISPOT) of the present invention in Embodiment 2.

FIG. 13 shows the results of detection of HBs antigen-specificantibody-secreting cells among human peripheral blood lymphocytes by themethod (FLISPOT) of the present invention in Embodiment 2.

FIG. 14 shows the results of Embodiment 3.

FIG. 15 shows the results of Embodiment 4 (the detection ofcytokine-producing cells).

FIG. 16 shows the results of Embodiment 6 (the application of functionalantibody to screening).

The invention claimed is:
 1. A method of screening for a target cell,comprising: causing specimen cells and a cell culture broth to becontained in at least a portion of the wells of a microwell arraycomprising multiple wells, each well being of a size permitting theentry of only a single cell therein, on one of the principal surfaces ofa base member, wherein a coating layer of a binding substance having theability to bind to at least a portion of a substance produced by atarget cell is present only around the wells of said principal surface;immersing the coating layer and the wells in the culture broth andculturing the cells in a state permitting the diffusion of saidsubstance produced by said target cell in the culture broth from thewells into the coating layer; after optionally removing the culturebroth, feeding a label substance binding specifically to said substanceproduced by said target cell present among the specimen cells onto thecoating layer; and detecting the binding of said substance produced bysaid target cell to said binding substance by means of said labelsubstance in order to specify the well containing said target cell. 2.The screening method according to claim 1, wherein the cells presentamong the specimen cells include cells that have been stimulated with adesired antigen in advance and are in a state capable of producing asubstance.
 3. The screening method according to claim 1, wherein thespecimen cells include immunoglobulin-producing cells orcytokine-producing cells.
 4. The screening method according to claim 1,wherein the specimen cells include immunoglobulin-producing cells, saidbinding substance is an anti-immunoglobulin antibody or antigen, and thetarget cell is an antigen-specific immunoglobulin-producing cell.
 5. Thescreening method according to claim 4, wherein the detection of thepresence or absence of the binding is conducted using antigen or anantibody to the immunoglobulin that is produced.
 6. The screening methodaccording to claim 1, wherein the specimen cells includecytokine-producing cells, said binding substance is an anti-cytokineantibody or a cytokine receptor, and the target cell is anantigen-specific cytokine-producing cell.
 7. The screening methodaccording to claim 6, wherein the detection of the presence or absenceof the binding is conducted using an antibody to the cytokine producedor a cytokine receptor.
 8. The screening method according to claim 4 or6, wherein the immunoglobulin-producing cells and cytokine-producingcells are natural cells, hybridomas, or cell strains.
 9. A method forobtaining a target cell producing a given substance, wherein said targetcell is from a group of cells containing specimen cells, comprising thesteps of: causing said specimen cells and a cell culture broth to becontained in at least a portion of wells of a microwell array, whereinsaid microwell array comprises multiple wells on a principal surface ofa base member, each well being of a size permitting the entry of only asingle cell therein, wherein a coating layer of a binding substancehaving the ability to bind to at least a portion of said substanceproduced by said target cell is bound on said principal surface, andwherein said binding substance is present only around the wells on saidprincipal surface; immersing the coating layer and the wells in theculture broth and culturing the cells in a state permitting thediffusion of said substance produced by said target cell in the culturebroth from the wells into the coating layer; after optionally removingthe culture broth, feeding a label substance binding specifically tosaid substance produced by said target cell present among the specimencells onto the coating layer; detecting the binding of said substanceproduced by said target cell to said binding substance by means of saidlabel substance in order to specify the well containing said targetcell; and recovering said target cell from a well thus specified. 10.The method according to claim 9, wherein said target cell is a specificimmunoglobulin-producing cell or specific cytokine-producing cell. 11.The method according to claim 9, wherein the cells present among thespecimen cells include cells that have been stimulated with a desiredantigen prior to introduction into the wells of said microarray and arein a state capable of producing a substance.
 12. The method according toclaim 9, wherein said target cell is an antigen-specificimmunoglobulin-producing cell and said binding substance is an antigenor an anti-immunoglobulin antibody.
 13. The method according to claim12, wherein said label substance is said specific antigen or ananti-immunoglobulin antibody.
 14. The method according to claim 9,wherein said target cell is an antigen-specific cytokine-producing celland said binding substance is an antibody to said cytokine, or acytokine receptor.
 15. The method according to claim 14, wherein saidlabel substance is an antibody to said cytokine, or a cytokine receptor.16. The method according to claim 10, wherein saidimmunoglobulin-producing cells or cytokine-producing cells are selectedfrom the group consisting of primary cells, hybridomas, and cell lines.17. The method according to claim 9, wherein the multiple wells arerecesses formed within the principal surface of the base member.
 18. Themethod according to claim 9, wherein said target cell is a mammaliancell.
 19. The method according to claim 9, wherein the wells have adiameter of 4 micrometers to 15 micrometers.
 20. The method according toclaim 9, wherein the wells have a diameter in a range of 0.5 to 2 timesa diameter of the cell.
 21. A method for obtaining a target cellproducing a given substance, wherein said target cell is from a group ofcells containing specimen cells, comprising the steps of: providing amicrowell array wherein said microwell array comprises multiple wellsand is formed on a principal surface of a base member, each well beingof a size permitting the entry of only a single cell therein, saidmicrowell array comprising a coating layer of a binding substance havingthe ability to bind to at least a portion of said substance produced bysaid target cell on said principal surface around the wells; causingsaid specimen cells and a cell culture broth to be contained in at leasta portion of the wells with the coating layer around the wells;immersing the coating layer and the wells in the culture broth andculturing the cells in a state permitting the diffusion of saidsubstance produced by said target cell in the culture broth from thewells into the coating layer; after optionally removing the culturebroth, feeding a label substance binding specifically to said substanceproduced by said target cell present among the specimen cells onto thecoating layer; detecting the binding of said substance produced by saidtarget cell to said binding substance by means of said label substancein order to specify the well containing said target cell; and recoveringsaid target cell from a well thus specified.